EP2663860B1 - Microstructure device for measuring molecular membranes and a method for producing said microstructure device - Google Patents

Description

The present invention relates to a microstructure device for measurement on molecular membranes, in particular bilipid layers, and to a process for producing this microstructure device.

Molecular membranes, in particular bilipid layers (double lipid layers), are used, for example, in membrane biophysics and cellular electrophysiology, as well as for certain nanoporous-based single-molecule analytical methods (molecular coulter counter). In these applications, in particular, the voltage-clamping technique ("voltage-clamp") is used to achieve a precise measurement of fluxes of charged particles (ions). The ion current is measured by means of a dielectric (insulating) separating layer (membrane) between two compartments containing electrolyte solution, which contains at least one ion-permeable pore or an ion channel. This membrane can be a bilipid layer, which is the typical basic constituent of natural biological (cell) membranes and can therefore be used as an artificial model of a natural cell membrane. For the voltage clamping technique, it is necessary to electrically contact two compartments, namely the electrolyte filled areas on either side of the membrane. For this purpose, the membrane is usually produced over the microaperture of a carrier substrate surface, wherein the microaperture forms the upper edge of a microcavity or a microhole in this carrier substrate. If the bilipid layer is applied over this surface, this molecular layer spans the microaperture "self-supporting" by means of its own surface tension. This one Microapertures appear "tail" in the optical microscope, they are also called "Black Lipid Membranes" (BLM).

Both for high precision of the measurement (high signal-to-noise ratio) and for a high throughput of voltage-clamp measurements, it is desirable to perform such measurements in microstructures in which the dielectric separation layer is integrated. The smaller the dimensions of the separating layer and the electrolyte-filled compartments, the lower the electrical measuring noise and the more such measuring arrangements can be accommodated in the form of an array on a small surface. A high density of such arrangements is a prerequisite for high throughput measurements.

All established devices for voltage-clamp measurements on dielectric separation layers or (cell) membranes are based on the structural principle that the membrane (cell membrane, synthetic lipid membrane, SiO 2 layer, SiN 3 layer, graphene layer or the like) a micro-aperture electrically possible tightly sealed, which is formed in an electrically insulating carrier layer, which separates two filled with electrically conductive medium (saline, electrolyte) compartments from each other. The electrical contacting, ie the transition from electronic to ionic conduction takes place in each case at one of two redox electrodes (typically Ag / AgCl) which in each case extend into one of the two compartments or are connected thereto via salt bridges. Typically, these are silver chloride coated silver wires with thicknesses between 0.5 and 2 mm. In order to bring these into electrical contact with the membrane relatively large, electrolyte-filled spaces are needed that require space and thus limit the integration density. In addition, a large surface wetted by electrolyte causes a high electrical capacity of the entire system, which has a negative effect on the measuring accuracy (parasitic capacitance) via the capacitive current noise.

A greatly simplified microstructure for voltage clamp measurements on membranes has therefore been proposed in each case Baaken, Prucker, Behrends, Rühe 2005, European Cells and Materials 5, Suppl. 5, p. CS4 ; Baaken, Prucker, Sondermann, Behrends, Rühe 2007, Tissue Engineering 18, 889 ; or in the document "Baaken et al." ( Baaken et al., "Planar microelectrode-cavity array for high-resolution and parallel electrical recording of membrane ionic currents", Lab Chip, 2008, 8, 938-944 ). In the microstructure described therein, in contrast to previous methods, the membrane was not applied on a microaperture between two compartments, but on the opening of a cavity introduced in an electrically insulating material (in the form of a blind hole). The electrical contacting of the few pL to several 100 fL large volume of electrolyte within the cavity is carried out in particular by means of a bottom of the same microgalvanically formed Ag / AgCI microelectrode (microelectrode cavity arrays, MECA). A similar microstructure device also describes the US 2009/0167288 A1 ,

This simplification reduces the space requirement per measuring position, which allows for much higher integration densities than before and also optimizes the electrical parameters (capacitance, access resistance); In addition, the production costs are reduced.

So far, however, this promising approach has a fundamental problem that seriously jeopardizes its practical suitability for high-throughput measurements and its reliability: in particular for the measurement of larger currents, as in measurements on whole cells (for a short time> 1 nA), as well as in measurements on nanopores (permanently> 100 pA), leash Ag / AgCl microelectrodes, in particular with diameters of <50 μm, are not satisfactorily stable. This is due to the fact that with small electrode areas very high current densities occur with a corresponding intensity of the chemical mass conversion at the electrode. As a result, after only a few minutes due to the redox reactions depending on the polarity of the electrode either an increasingly thick, poorly conductive AgCI layer (When switching as an anode) or the AgCl degraded so far that only reduced Ag is present (when switched as a cathode). In the first case, the resistance of the electrode increases massively, in the second only more capacitive coupling to the electrolyte in the manner of a polarizable electrode is possible and the DC resistance increases abruptly. It would be desirable to have a constant, drift-poor electrical behavior of the electrode.

The publication SD Ogier et al "Suspended Planar Phospholipid Bilayers on Micromachined Supports", Langmuir, Vol. 16, No. 13, June 1, 2000, pp. 5696-5701 describes a device as mentioned in the preamble of claim 1.

It is an object of the present invention to provide an improved microstructure device and a method for the production thereof, the electrical measurement properties in the electrolyte are particularly stable as long as possible when measured on a molecular membrane.

This object is achieved by the microstructure device according to claim 1 and the method according to claim 7. Preferred embodiments of the method and the device used in this are subject of the dependent claims 2 to 6 and 8.

If one uses the microstructure device according to the invention, for example for measurement on a molecular membrane by means of voltage clamping, as described above, it results that the current flowing through the electrode and membrane current at the electrode causes a relatively low current density, since the contact side with the electrolyte has a relatively large characteristic Diameter. The relatively low electrical current density (current / area) of the contact side has the advantage that the per-area electrochemically induced reactions are reduced at the contact side of the electrode, which reorganize the electrode over time and thus change their properties. In this way, especially when using a Silver / silver chloride electrode, the formation of the disadvantageously thick silver chloride layer (anode connection of the electrode) or the adverse total regression of the silver chloride layer (cathode circuit of the electrode) are delayed. As a result, the electrode has more constant electrical properties, in particular a lower drift and a longer measurement duration (before the electrode can be restored, if necessary, at least partially by reversing the polarity).

In the present case, the characteristic diameter of a side or surface is understood to mean the diameter of a circular surface which has the same absolute size as this side or surface. For example, the characteristic diameter D of a square having the side length a is D = 2 * a / sqrt (pi) and the characteristic diameter D of a circle having the radius D / 2 is D.

The direction designation "top" or "upward" in the present case denotes the direction of the normal vector which points away from the microcavity plane from the plane of the microaperture. This direction is also defined as the direction of the positive z-axis of a Cartesian coordinate system (the origin of this coordinate system is preferably not determined hereby). The direction designation "down" or "down" designates in the present case the direction of the normal vector pointing into the microcavity from the plane of the microaperture (also referred to as the direction of the negative z-axis of this coordinate system). The plane of the microaperture is the plane that is coplanar with the surface framed by a planar microaperture (microaperture surface). The microaperture preferably lies substantially completely in a plane which runs in particular parallel to the x-y plane of this coordinate system. However, the microaperture can also run in more than one plane, in particular have a continuous (stepless) course.

The characteristic diameter D2 of the contact side of the electrode is preferably greater than D1 * c (D1 multiplied by a factor c), wherein each c is preferably: 1.1; 1.2; 1.3; 1.4; 1.5; 2.0; 3; 4; 5; 10; 15; 20; 25, 35 or 50. The ratio D2 / D1 can also be larger. Even at a ratio of D2 / D1 = 1.1, an improvement in the stability of the electrode compared to the case D2 / D1 = 1.0 to be watched. However, in particular when D 2 is at least 1.5 times greater than D1 (D 2> 1.5 * D 1) and, preferably, smaller than 20 (D 2 <20), it is preferred. This results in particularly suitable electrode properties in the case of photolithographic means-easily produced microstructures. The most stable electrodes were found to be 2.0 <D2 / D1 <20. If D1 is kept constant, then at ratios D2 / D1> 20 it must be accepted that the area required for the individual electrode and measuring point becomes relatively large, so that the possible density of the electrodes per carrier substrate surface decreases. Nevertheless, even such large ratios D2 / D1 can be advantageous, in particular with regard to the stability of the electrode properties.

In principle, D2 / D1 = 1 could also be used. It would, however, e.g. However, a simple increase in the diameter of the microcavity and thus both the electrode forming the bottom of the same and the microaperture reduce the current densities and improve the electrode stability would not solve the problem satisfactorily, since small microaperture diameters are to be preferred in particular for high-resolution measurements on nanopores ( In particular <50 microns) and small Mikroaperturdurchmesser (eg <10 microns) are particularly preferred for measurements on cells.

Preferably, the carrier substrate has a first layer, within which the electrode is at least partially or substantially completely arranged. A layer may be a layer in the carrier substrate or a coating of the carrier substrate. Such a layer is preferably at least partially or completely substantially planar. If the electrode is arranged completely in the first layer, this preferably means that no portion of the electrode protrudes from the first layer or does not penetrate the imaginary main planes, which envelop the layer, for example, upwards and downwards. The carrier substrate may comprise at least one layer, in particular more than two layers or a plurality of layers, wherein the microcavity may be arranged in one of these layers, several of these layers or each of these layers or may not be arranged.

The first and / or second layer of the carrier substrate is preferably a coating of the carrier substrate, which preferably forms its upper side and is preferably hydrophobic, preferably consists of a photosensitive layer, in particular an epoxy resin or photoresist, or has these. The coating preferably consists of a polymer, preferably of epoxy resin or preferably of a photoresist, e.g. SU8 or has such a material. This photoresist is preferably SU8, the dried layers of which are hydrophobic. SU-8 is a commercially available photoresist from Microchem Corp., USA, and belongs to the group of negative resists. Like most resists, SU-8 consists of the three components base resin, solvent and photosensitive component. These are particularly suitable for the production of the microstructure device, since in particular many Bilipidschichten form particularly reliable on these resist and since they are photolithographically processed, for. Microstructures produce, and are chemically relatively inert.

The coating of the carrier substrate may further preferably comprise polytetrafluoroethylene (PTFE) or consist of this material. The advantage is that such layers are particularly chemically inert, making them particularly suitable for use in corrosive environments (e.g., physiological electrolyte-physiological saline solutions).

Preferably, the microstructure device has at least one feed device, by means of which the electrode is electrically contactable via a feed line distance. Preferably, one (or each) electrode is assigned at least one supply device. A feeder device may comprise or consist of a metallic conductor, which may consist for example of gold, titanium, nickel-chromium, platinum, or silver or has one or more of these materials. In order to distinguish between electrode and supply device, it can be defined that the parts of the system of electrode and supply device which are in contact with the electrolyte or with the inner volume of the microcavity are attributed to the electrode while they are not in contact with the electrolyte or with the internal volume of the microcavity standing parts of the system of electrode and supply of the Zuleitungseinrichtung be attributed. The supply line device is preferably arranged completely or at least partly in the first layer.

The electrode preferably forms at least part of the inner wall of the microcavity. The microcavity is preferably designed such that the inner volume of the microcavity is limited by the area framed by the microaperture and the inner wall (or several inner walls) of the microcavity. The at least one inner wall of the microaperture is preferably formed from lateral inner walls and a bottom wall. The bottom wall of the microcavity is preferably formed completely or at least partially from the contact side of the electrode. However, the electrode can also be at least partially or completely surrounded by the internal volume of the microcavity. For example, the electrode may be partially self-supporting.

Preferably, this first layer is disposed substantially below or at least partially below the microcavity. This makes possible, in particular, the simple production of the electrode, which is preferably arranged below the microcavity.

Preferably, the carrier substrate has a second layer within which the microcavity is at least partially or substantially completely disposed. This makes possible, in particular, the simple production of the microcavity, which is preferably arranged above the electrode. However, it is also preferred that the microcavity is at least partially, preferably with less than half or a quarter of its internal volume, also arranged in the first layer.

It has the microcavity a second, upper, space portion which is bounded by the Mikroaperturfläche and (at least at a level or in the average of all heights) in cross-section has a characteristic diameter D3. Further, the microcavity has a first, lower, space portion located below the upper space portion and having a characteristic diameter D4 (at least at a height or average of all heights) in this cross section, where D4 is greater than D3 (D4> D3 in particular D4 is greater than D3 * c (D3 multiplied by a factor c), where c is in each case preferably: 1.5; 2.0; 3; 4; 5; 10; 15; 20; 25; 50, and further preferably substantially D4 = D2 and substantially D3 = D1 (the considered cross-section passes through the geometric center of a planar microaperture surface and is perpendicular to this). A space portion is preferably substantially cylindrical, wherein the cylinder axis is preferably parallel to the z-axis. However, a space portion may also be substantially cuboid or otherwise formed.

The characteristic height H3 of the second space section is preferably between 1 .mu.m and 50 .mu.m, preferably between 1 .mu.m and 10 .mu.m or between 3 .mu.m and 6 .mu.m, but may also be dimensioned differently. The characteristic height H4 of the first space section is preferably between 0 μm and 50 μm, preferably between 0 μm and 10 μm, between 3 μm and 6 μm, particularly preferably between 0.0005 μm and 1.0 μm or 0.0005 μm and 0 , 5 μm or between 0.0005 μm and 0.05 μm or between 0.001 μm and 0.05 μm, but may also be different. H4 is smaller than H3. The lower space portion is preferably for contacting the relatively large contact side of the electrode having a characteristic diameter D2 by the electrolyte. To this end, if possible, the entire contact side of the electrode should be contactable by preferably substantially D4 = D2, while the height H4 of the lower space portion for the electrical contacting of the contact side by the electrolyte need not be large. Accordingly, H4 can be chosen small. As a result, at small values H4, smaller distances of the electrode from the microaperture, which is intended to carry the membrane to be measured, result. As a result, in particular when used for the voltage clamping technique, it is possible to improve the signal-to-noise ratio. Furthermore, the microstructure device can be kept compact especially at low values H4. Preferably, the first or lower space portion is completely within the first

Layer arranged and preferably the second or upper space portion is disposed completely within the second layer. Thereby, a space portion can be easily fabricated by removing it by photolithographic means (particularly by the use of a mask and UV light) from the layer, which preferably consists of a photoresist (e.g., SU8).

The "characteristic height" H4 (or H3) of the space portion is preferably an average value. It results preferably from the fact that a fictitious (cylinder) volume V is assigned to the space section, which results from the product of a characteristic circular area A and this characteristic height H4 (V = A * H4 or V = A * H3). Apart from that, a space portion may actually be cylindrical. The characteristic circular area A results from the characteristic diameter D4 (or D3) (A = (D4 / 2) ^ 2 * pi). The characteristic diameter of the space portion is, in particular at constant lateral dimension or constant diameters of the space portion, preferably at least considered in a height or, especially with variable lateral dimensions or depending on its height variable diameters of the space portion, preferably as an average of the characteristic diameter in considered all heights of this room section. For example, the characteristic diameter Dk of a rotationally symmetric cone having a diameter D of the circle base and a height h is Dk = D / 4, and the characteristic diameter Dz of a cylinder having a diameter D of the circular base of the cylinder is Dz = D.

In particular, a contiguous internal volume of the microcavity is considered as the space section, which can be filled in particular with water or an aqueous electrolyte. It has surprisingly been found in experiments that especially low or very low heights H4 (eg 0.0005 μm <H4 <0.5 μm) are sufficient to electrically contact an electrode with a relatively large characteristic diameter D2> D1. This has been achieved in particular by such volumes of the second space section, which are defined in particular by an open-pore or sponge-like filling material in that the volume is formed by the contiguous internal volume of the pores. The second room section can be defined by an open-pore or sponge-like filling material. This volume of the second space portion may be framed, for example, by the electrode and by inner wall portions defined by the carrier substrate which may be cylindrical, and by the underside of the first space portion.

The contact side of the electrode preferably has a non-planar contact interface with the inner volume of the microcavity, at least in one section (or everywhere), and in particular on a microscopic or nanoscopic scale, this contact interface being larger than the surface of the macroscopically planar contact side in this section. This can be achieved by elevations (strip-shaped, spot-shaped, plateau-like) or depressions (channel-like, hole-like, channel-like) of the contact side. It can also be achieved by microstructuring (characteristic distances of the surface structures in the range of 1 .mu.m to 1000 .mu.m) or nanostructuring (characteristic distances of the surface structures in the range of 0.5 nm to 1000 nm) of the contact side of the electrode or by a material property of the contact side (eg Roughness, porosity, especially microporosity or nanoporosity, as in Ag / AgCl electrode). By enlarging the electrode interface, in operation of the electrode, the electric current density (electric current / electrode interface) at the electrode can be reduced and the electrode can be made more stable, e.g. in a voltage clamping technique blade assembly of the microstructure device.

It has also been found that, in particular, electrochemically produced electrodes made of the material silver / silver chloride (Ag / AgCl) have a nanoporous layer as a typical property, which forms a coherent internal volume, which is filled in particular by an electrolyte arranged in the microcavity. The nanoporous layer can thus be assigned a second space section with characteristic height H4 and characteristic diameter D4. The nanoporous layer consists of nanoporous AgCl or AgCl. Typical grain sizes of the nanoporous layer are 50 nm to 500 nm. An Ag / AgCl electrode is preferably formed by electrochemical deposition from a silver nitrate solution. Further details can be found eg in the document "Baaken et al.". The porosity is one Standard result of microplating, during which the silver is electrochemically deposited on a gold starting layer, for example. The porosity depends on the electrical parameters (current, voltage), duration and concentration of the electrolytes used (silver nitrate solutions). With slight changes, for example, higher concentrations of the silver nitrate solution, the deposited "silver crystals" become significantly larger.

It should be noted that the volume and the height of the second space portion will change in operation of the microstructure device when the microstructure device has an electrode that changes in the electrolyte depending on the electrical polarity of the electrode. This is the case with Ag / AgCl electrodes, as explained above. However, since the nanoporous AgCl, which increases or decreases on the electrode as a function of their polarity, is sufficiently open-pore nanoporous, ie forms a sufficiently coherent internal volume, essentially the entire electrode surface with characteristic diameter D2 remains electrically contacted even if an original one eg hollow cylinder-like second space section is later filled substantially completely by the nanoporous material.

The carrier substrate is preferably formed at least in sections or substantially completely planar. The carrier substrate has one or more microstructures, that is, spatial highlights and / or pits with small dimensions, e.g. these microcavities e.g. a few nanometers, a few micrometers, a few tens of micrometers or a few hundred micrometers. Such microstructures can be e.g. produced by known optical lithographic processes in which structures defined by means of optical masks are applied in layers on a carrier substrate and partially removed again.

The carrier substrate preferably has an upper side, which is preferably at least partially or substantially completely planar. The upper side preferably has at least one microaperture, preferably a number N of microapertures, where N is in each case preferably between 2 and 2000, greater than 2000, preferably between 2 and 400, between 4 and 100, between 4 and 50, or between 4 and 20. Via a microaperture, a "cantilevered" molecular membrane (BLM), for example a bilipid membrane, can be produced. This is done, for example, by "brushing"("paintingmethod","painting") a solution of the first solvent (eg hexane, heptane, octane, nonane, decane, hexadecane or other alkanes, or a mixture of one or more of these substances) and this lipid at a concentration of eg 1 mg / ml on the carrier substrate over this microaperture. The self-supporting molecular layer then separates two compartments from each other, which allows measurement arrangements for the realization of a voltage clamping technique. The use of multiple microapertures and microcavities has the advantage that several such sensors can be operated in parallel, which allows a higher measurement throughput.

Such a molecular membrane may comprise or consist of a layer of amphiphilic molecules, in particular lipids. The layer may, in particular, be a double layer, that is to say consist of two individual layers arranged one above the other, wherein a single layer consists in particular of self-assembled molecules. Such a molecular membrane may be made artificially, in particular a bilipid layer of lipid molecules may be prepared by means of painting, vesicle fusion or Langmuir-Blodgett / Langmuir-Schäfer technique. Such artificial lipid membranes are often used as models of natural membranes. They serve, for example, as an environment for the investigation of membrane proteins which, for example, transport charges through the membrane between two compartments separated by the membrane. However, such a membrane can also be a natural, biological membrane which is applied over the at least one microaperture. This can be done by using a substantially planar patch of the membrane of a biological cell or by using a complete, treated or untreated biological cell which rests on the at least one microaperture. The thickness of the molecular layer in particular has molecular dimensions, in particular between 1 nm and 100 nm.

A microaperture is understood to mean the open cross-section, e.g. through an opening, e.g. a recess or a hole, in a -particularly planar-surface of the top of the carrier substrate results. The shape of the microaperture contour is preferably circular, ellipsoidal, triangular, quadrangular or polygonal. The maximum, minimum or average diameter of the individual microaperture is preferably less than 1000 μm and is preferably between 500 nm and 500 μm, preferably between 2 μm and 250 μm, preferably between 2 μm and 50 μm, preferably between 2 μm and 10 μm. or between 5 μm and 150 μm. In such preferred Mikroaperturgrößen (microapertures), a molecular layer over the microaperture can be generated, which is not possible in macroscopic apertures with diameters of several millimeters in the rule.

Such microapertures may be formed by selectively removing a photosensitive layer, eg, photoresist, mounted on the top of the support by optical lithography, such as the document "Baaken et al." or the US 2009/0167288 A1 described. However, the microaperture may also form the edge of a hole extending from the top to the back of the carrier substrate. This can be achieved, for example, by chemical etching or by irradiation with laser or other high-energy radiation.

The arrangement of the number N of microapertures preferably corresponds to an array, preferably a periodic lattice, in which the position of the microapertures or the microaperture centers can be described by one or a few lattice parameters. The arrangement in a periodic grating has advantages in the design of a parallelized sensor system in which many identical measuring points are to be created. However, the microapertures may also be arranged in a non-periodic or incompletely periodic pattern.

The carrier substrate is preferably made of glass or has glass. However, it can also consist of a semiconductor material or have this at least, for example Si / SiO 2. Other materials are also possible. The top of the carrier substrate preferably has a coating. This is preferably hydrophobic, but may also be hydrophilic. The advantage of a hydrophobic top surface is that many types of bilipid layers form on such surfaces particularly reliably.

In the context of the present invention, a "hydrophobic" boundary layer is understood to mean a layer on which a water droplet has a contact angle of at least 70 °, preferably at least 80 °, 85 ° or 90 °, preferably between 80 ° and 130 ° or between 90 ° and 120 °. As such, the present definition of the term "hydrophobic" may be broader than is commonly used in the literature, where the term most often refers to contact angles greater than 90 °. Such contact angles (interior angle of the water drop on the substrate) can be easily determined by means of commercially available contact angle measuring devices or by evaluation of light microscopic cross-sectional images of the drops (room temperature, standard conditions). In a hydrophilic barrier layer, the contact angles are each preferably between 70 ° and 0 °, 80 ° and 0 °, 85 ° and 0 ° or 90 ° and 0 °.

Preferably, a carrier substrate has at least one microcavity, or preferably an array of microcavities, one or each microcavity being open at the top and terminating in one of said microapertures in the top of the carrier substrate. The molecular membrane to be measured can then be formed such that it covers at least one microaperture or several microapertures. A microcavity is a depression in the top whose depth can be the same size as a possible called microaperture diameter, or can be deeper or less deep. Each cross section through the depression (in a plane parallel to the plane of the microaperture) preferably has the same cross section as the microaperture, which opens the microcavity upwards. The microcavity can in particular be cylinder-like or cuboidal. But it can also be hollow cone (dull) shaped or have another shape with variable cross-section.

The microstructure device is preferably designed as a measuring arrangement or is part of a measuring arrangement. Such a measuring arrangement preferably has the Microstructure device on and at least a first compartment, namely a space area or a chamber. A microcavity can serve as a "second compartment" of a measuring arrangement. The microstructure device further preferably has at least one wall section, which is arranged above the carrier substrate and defines with the carrier substrate or its coating (s) the chamber or the first compartment in which a liquid volume of a few microliters and preferably up to one milliliter can be arranged is. Thus, the microstructure device may have a chamber portion for receiving such a liquid volume, in particular a first and a second solvent (electrolyte). The first and the second compartment are connected via the at least one microaperture. The compartments are separable, in particular electrically separable, by arranging a membrane above the microaperture, for example a bilipid layer. This membrane separates the electrolyte-filled compartments electrically tightly from each other. An electrolyte for contacting the underside of a molecular layer arranged on the microaperture can preferably be arranged or arranged within the second compartment. In the microcavity, the electrode is preferably arranged, by means of which the electrolyte in the second compartment is electrically contacted (corresponding to the arrangement in FIG Fig. 1 ). Furthermore, at least one counterelectrode is preferably arranged in the first compartment, wherein in this first compartment an electrolyte can be arranged or arranged. With this measuring arrangement, which has the microstructure device, a voltage clamping technique measuring arrangement can be realized.

The measuring arrangement or the microstructure device preferably has at least one sensor device, which in particular has a sensor for electrophysiological examinations on the molecular layer, in particular bilipid layer. The sensor device may comprise this electrode in the second compartment, which is arranged on this side of the microaperture on the carrier substrate, and may further comprise at least one further electrode (counter electrode) on the other side of the microaperture, which is arranged in the first compartment in the electrolyte above the molecular layer. Such an electrode is preferably a redox electrode, preferably a "second type" redox electrode, eg an Ag / AgCl electrode or a calomel electrode. The electrode is preferably a non-polarizable electrode, which allows a simple transition of the ionic charge carriers in the electrolyte into electronic charge carriers in the metal. Ag / AgCl electrodes are preferably used for this purpose, preferably in combination with chlorine ion-containing measurement solutions (electrolyte).

This sensor device is preferably designed for carrying out the voltage clamping technique, by means of which, with the voltage kept constant, the smallest currents in the nanoampere range and below can be measured, in particular in the picoampere range, e.g. using a voltage-clamp amplifier or a patch-clamp amplifier (e.g., an Axopatch 200B, Axon Instruments, Foster City, CA operated in resistive feedback mode). The sensor device may comprise an array of sensors, which may be arranged in the carrier substrate or on its surface.

As a solvent, namely as an electrolyte, which is placed below and / or above the membrane (molecular layer), in particular saline solutions come into question, in particular physiological saline solutions, the electrophysiological measurement of Molkülschicht, eg lipid membrane, and therein, charge-transporting pores, eg channel proteins. Suitable solvents, in particular for carrying out measurements by means of voltage clamping technology, result, for example, from the document "Baaken et al." or the US 2009/0167288 A1 ,

Suitable amphiphilic molecules for the preparation of the molecular layers are, in particular, lipids, in particular lipids suitable for the formation of membrane-like double lipid layers, as described, for example, in the document "Baaken et al." or in US 2009/0167288 A1 to be named.

Preferred embodiments of the device according to the invention can also be taken from the description of the method according to the invention and vice versa.

The method according to the invention for the production of a microstructure device according to the invention has at least the steps, in particular without necessarily stipulating the order of the steps in this case: provision of a carrier substrate; - applying at least one electrode to the carrier substrate; - applying a (structurable) layer above the carrier substrate; Forming the at least one microcavity by selectively removing portions of that (structurable) layer, which portions at least partially or completely form the microcavity.

Preferably, the method comprises the step of forming this (structurable) layer directly on the carrier substrate or on a layer of the carrier substrate. Alternatively, the method comprises the step of forming this structurable layer indirectly on the carrier substrate by first forming this structurable layer on a second, separate carrier substrate and then transferring it from this second carrier substrate to the first carrier substrate or a layer on the second carrier substrate is (preferably by "bonding", as explained in the document "Baaken et al.").

Further properties and features of the microstructure device and for the production of the microstructure device, materials, methods, measuring arrangements and examples for measurement on membranes can be found in the document "Baaken et al." or the US 2009/0167288 A1 be removed

• Fig. 1 zeigt schematisch ein erstes Ausführungsbeispiel der erfindungsgemäßen Mikrostrukturvorrichtung zur Messung an einer Membran in einem schematischen, senkrechten Querschnitt.
• Fig. 2a zeigt ein zweites Ausführungsbeispiel der erfindungsgemäßen Mikrostrukturvorrichtung als schematischen, senkrechten Querschnitt.
• Fig. 2b zeigt die Mikrostrukturvorrichtung der Fig. 2a als schematische Aufsicht.
• Fig. 3a zeigt ein drittes Ausführungsbeispiel der erfindungsgemäßen Mikrostrukturvorrichtung als schematischen, senkrechten Querschnitt.
• Fig. 3b zeigt die Mikrostrukturvorrichtung der Fig. 3a als schematische Aufsicht.
• Fig. 4 zeigt eine mit einer erfindungsgemäßen Mikrostrukturvorrichtung gemessene Spannungskurve V(t).
• Fig. 5 zeigt eine mit einer erfindungsgemäßen Mikrostrukturvorrichtung gemessene Stromkurve I(t).
• Fig. 6 zeigt schematisch zwei Ausführungsbeispiele des erfindungsgemäßen Verfahrens zur Herstellung der erfindungsgemäßen Mikrostrukturvorrichtung.

Further preferred embodiments of the method according to the invention and the microstructure device according to the invention will become apparent from the following description of the embodiments in conjunction with the figures. Like reference numerals denote substantially the same components.

• Fig. 1 schematically shows a first embodiment of the microstructure device according to the invention for measurement on a membrane in a schematic, vertical cross-section.
• Fig. 2a shows a second embodiment of the microstructure device according to the invention as a schematic, vertical cross section.
• Fig. 2b shows the microstructure device of Fig. 2a as a schematic view.
• Fig. 3a shows a third embodiment of the microstructure device according to the invention as a schematic, vertical cross section.
• Fig. 3b shows the microstructure device of Fig. 3a as a schematic view.
• Fig. 4 shows a measured with a microstructure device according to the invention voltage V (t).
• Fig. 5 shows a measured with a microstructure device according to the invention current curve I (t).
• Fig. 6 schematically shows two embodiments of the inventive method for producing the microstructure device according to the invention.

FIG. 1 shows the microstructure device 1, which is designed as a measuring arrangement for electrophysiological measurement on a membrane 11, namely a Bilipidmembran, by means of stress-clamping technology. The microstructure device 1 has a carrier substrate 2 made of glass, on which a first layer 3 of photoresist (SU8) was applied by spin-coating. The first layer 3 has a hollow cylindrical recess 5, which was produced in particular by a photolithographic method within which a redox electrode 6 is mounted on the upper side of the carrier substrate 2. Above the first layer 3, a second layer 4 of photoresist (SU8) is applied, which was produced by spin-coating the liquid photoresist on the first layer 3. The method step of producing the second layer of epoxy resin on a first layer of epoxy resin by spin coating, wherein the first layer is in particular already microstructured, namely in particular already has the recesses 5, has proved to be surprisingly successful exposed. The second layer is fixed, that is not non-destructively separable, connected to the first layer and forms a planar top 10 of the microstructure device or of the extended by the layers 3, 4 support substrate 2, which carries the membrane 11. The second layer 4 has a hollow cylindrical recess 7, which forms an internal volume with the recess 5 within the composite layer of first layer 3 and second layer 4. The inner volume of the microcavity 8 is formed by the first space portion defined by the recess 5 through the second space portion defined by the recess 7. The inner walls of the recesses 5 and 7 in the layers 3 and 4, a portion 13 of the underside of the second layer and the bottom contact side 12 of the electrode 6 define the microcavity 8 and serve as the first compartment 8 and the liquid electrolyte chamber 8, respectively ,

The upwardly open microcavity 8 opens at its upper side into the microaperture 9, which is arranged in the planar upper side 10 of the microstructure device and which has a circular area of the microaperture 9 with the diameter D1. D1 is the characteristic diameter of the microaperture 9.

The electrode 6 has the shape of a flat cylinder and is arranged in a form-fitting manner in the region of the recess 5 in the microcavity 8. Its contact side 12 adjoins the internal volume of the microcavity 8 in order to enable electrical contacting of an electrolyte arranged in the microcavity. The contact side 12 of the electrode 6 has a diameter D2. D2 is the characteristic diameter of the contact side 12 of the electrode 6. D2 is about three times as large as D1. Using the microstructure device 1 for measurement on a molecular membrane 11 by means of voltage clamping technique, it follows that the current flowing through the electrode and membrane at the electrode 6 causes a relatively low current density, since the contact side 12 with the electrolyte has a relatively large characteristic diameter D2 having. The relatively low electrical current density (current / area) of the contact side 12 has the advantage that the per-area electrochemically induced reactions are reduced at the contact side of the electrode, which reorganize the electrode 6 over time and thus change their properties. This indicates the electrode more constant electrical properties, in particular a lower drift and a longer measurement time.

The defined by the first recess 5 and the contact side 12 first space portion is hollow cylindrical and has a diameter D4. D4 is the characteristic diameter of the first space section of the microcavity 8. The second space section defined by the second recess 7 is also hollow cylindrical and has a diameter D3. D3 is the characteristic diameter of the first space portion of the microcavity 8. Due to the geometry chosen to form the microcavity 8, D1 = D3 and D2 = D4. The first space section has a height H4. H4 is the characteristic height of the first space portion of the microcavity 8. The second space portion has a height H3. H3 is the characteristic height of the second space portion of the microcavity 8. Due to the geometry chosen to form the microcavity 8, H4 <H3. In particular, such relatively small values for H4 have been proven to reliably contact the electrode 6 with the electrolyte in the microcavity 8.

Above the upper side 10 of the carrier substrate 2 form wall sections 14, the second compartment 15, which forms a chamber for liquid-tight receiving an electrolyte. The first compartment 8 and the second compartment 15 are electrically in the region of the microaperture 9 is separated by the membrane 11, which rests tightly on the microaperture 9. By means of a counterelectrode 16, which is arranged in the electrolyte of the second compartment 15, it is now possible, with the further use of measuring electronics 17, to realize a voltage-clamping technique measuring arrangement. The microcavity 8 is closed at the bottom by the inner redox electrode 6 (eg Ag / AgCl). This is electrically connected to the sensor electronics 17 of the sensor device via, for example, a lithographically generated supply line 18. In the electrolyte of the chamber 15 is the redox electrode 16, which serves as a reference electrode. By voltage clamping technique, in which an electrical voltage across the membrane is kept constant, for example by means of a patch-clamp amplifier, least current changes of a stream of charges "q" can be detected through the membrane, which spans the microaperture 9 as BLM. These charges can be on the Ion transport due to leaks in the cell membrane or based on pores, eg channel proteins in the cell membrane go back.

FIG. 2a shows a second embodiment of the microstructure device 20. The microstructure device 20 has a carrier substrate (not shown) on which the first layer 23 of SU8 photoresist was applied by spin-coating on the carrier substrate. The first layer 23 has a hollow cylindrical recess 25, which was produced by photolithography. Within the recess 25, the electrode 26 is fitted. The electrode 26 consists of several layers. It has a first layer 27 of gold which has been photolithographically produced and which contacts and is bonded to the glass substrate (not shown). This layer serves as a starter layer for applying a layer 28 of silver, for example by vapor deposition. Starting from the layer 28 of silver, the layer 29 of silver chloride is produced on the layer 28 of silver by electrochemical deposition of silver chloride from a silver nitrate solution. In this way, a silver / silver chloride electrode 26, which serves as a redox electrode for electrically contacting a chloride ion-containing electrolyte, is obtained in the recess 25.
Above the first layer 23, the second layer 24 of photoresist SU8 is spin-coated. In the second layer 24, the recess 30 is arranged, which opens into the microaperture 31 in the upper side 32 of the extended by the layers 23, 24 support substrate, which serves to support the membrane.

The inner volume of the microcavity of the microstructure device 20 is formed by the first space portion defined by the first recess 25 in the first layer 23 and the second space portion defined by the second recess 30 in the second layer 24. This is surprising in that the first recess 25 is substantially completely filled by the electrode 26. However, it has surprisingly been found that the coherent cavity or contiguous cavities between the pores of the nanoporous silver chloride layer 29, which are connected to the second space portion 30, define a first space portion suitable for electrically contacting the electrode 26 with a liquid electrolyte. This first room section can be characteristic diameter D4 and a characteristic height H4, which are different from the corresponding real dimensions of the silver chloride layer 29, namely smaller. The silver chloride layer 29 has a composition of grains having a grain size of typically 50 nm to 500 nm. The internal volume of their interstices forms the volume of this first space section. Depending on the polarity of the electrode 26 during operation of the microstructure device, the thickness of the silver chloride layer 29 may change, in particular in a silver chloride-containing electrolyte, so that the volume of the first spatial section can increase and decrease. It is H4 <H3.

Typical dimensions of the microstructure device 20 may be e.g. be selected as follows: characteristic diameter D1 of the microaperture = diameter of the microaperture and characteristic diameter D3 of the second spatial section = diameter of the second spatial section: D1 = D3 = 3 μm-20 μm; Characteristic diameter D2 of the electrode = diameter of the electrode and characteristic diameter D4 of the first space portion = diameter of the first space portion: D2 = D4 = 10 μm - 100 μm, e.g. 60 μm; Total height h5 of the composite layer of first layer 23 and second layer 24: h5 = 14-20 μm; Total height h2 of electrode 26: h2 = 7 μm - 10 μm; Height h1 of the layer 27 of the electrode: about 0.2 μm; Height of the layer 28 of the electrode: 6 μm - 9 μm; Height of silver chloride layer 29: varying, 10 nm to 2 μm.

Fig. 3a shows as another embodiment, the microstructure device 40, which is similar to the microstructure device 20 and has been prepared analogously. The microstructure device 40 has a carrier substrate (not shown) on which the first layer 43 of SU8 photoresist has been applied. The first layer 43 has a hollow cylindrical recess 45. Within the recess 45, the electrode 46 is fitted. The electrode 46 consists of several layers. It has a first layer 47 of gold and contacts and is bonded to the glass substrate (not shown). This layer serves as a starter layer for applying a layer 48 of silver. Starting from the layer 48 of silver, the layer 49 of silver chloride is formed on the layer 48 of silver. In this way you get in the Recess 45 a silver / silver chloride electrode 46, which serves as a redox electrode for electrically contacting a chloride ion-containing electrolyte.

Above the first layer 43, the second layer 44 of photoresist SU8 is ejected. In the second layer 44, the recess 50 is arranged, which opens into the microaperture 51 in the upper side 52 of the extended by the layers 43, 44 support substrate, which serves to support the membrane.

The inner volume of the microcavity of the microstructure device 40 is formed by the first space portion defined by the first recess 45 in the first layer 43 and the second space portion defined by the second recess 50 in the second layer 44. The second space portion, in contrast to the microstructure device 20, is produced not only by the nanoporosity of the silver chloride layer 49, which can be assigned a characteristic height h6, but also by the free, hollow cylinder-like portion 53 of the recess 45, which is between the contact side facing the internal volume of the microcavity the electrode 46 and portions 54 of the underside of the second layer 44 is formed. This section has a height h7. The second space section thus has a characteristic height H4, which corresponds to the real height h7 of the free section 53 plus the characteristic height h6 of the silver chloride layer 49. By the free portion 53, the contact side of the electrode 46 for the electrolyte is even more accessible. It is H4 <H3.

In both types of microstructure device 20 and 40, the stability of the electrodes could be significantly improved by the relatively large electrode area (D2> D1) and, in particular, by the provision of a significantly higher amount of nanoporous AgCl material compared to smaller electrodes.

Fig. 4 shows a voltage curve V (t) measured with a microstructure device 20 or 40 (with Ag / AgCl electrodes (D2 = 50 μm)). This is a stability test of the Ag / AgCl electrodes embedded in an insulator layer (SU8). The graphs describe the course of the voltage required for a constant current of + -10 nA over time.

The electrode was switched for 3 cycles as a cathode or as an anode and each operated until the loss of stability. With the electrodes thus obtained, it has already been possible to carry out the first very high quality measurements on bilipid layers containing alamethicin (see Fig. 5 ) or botulinum toxin C2. This showed that the expected improvement of the electrical parameters was fully realized.

It is in Fig. 4 the voltage required for impressing a current of +/- 10 nA over time. The electrode was switched cyclically, between polarity as anode and as cathode. In one case, a silver chloride layer grows on the surface, which after a certain time, due to the high electrical resistance leads to a significant increase in the voltage required for a current of 10 nA. At this point, the test setup was reversed, ie the microelectrode switched as the cathode. Silver chloride is now reduced to silver on the surface. If silver chloride is no longer present, the resistance and the voltage increase strongly again. The electrode is no longer stable and has been reversed again and so on. In total, 3 cycles (back and forth) were run.

It can be seen that the electrodes, irrespective of whether connected as a cathode or an anode, can keep the current in the required voltage for more than half an hour without a significant deviation. It could thus be shown that the stability criteria for the measurements on cells or on highly conductive bacterial pores are more than fulfilled. If one uses a current of 1 nA, for example, the time available for the measurements in a polarization direction is increased to, for example, about 6 hours. Remarkably, after the loss of stability in one direction of polarization, the electrodes can be completely restored when operating with the opposite driving force, as can the passage of several cycles in Fig. 4 clarified.

Fig. 5 shows a measured with a microstructure device 20 or 40 current curve I (t). These are typical current waveforms for voltage clamping of alamethicin-mediated currents in a lipid bilayer. Noteworthy are the exceptionally high seal resistances (several 100 GOhm), which on the SU8 chips the microstructure devices 20, 40 were obtained. This significantly improves the signal-to-noise ratio again.

Fig. 6 schematically shows two embodiments of the inventive method for producing the microstructure device according to the invention, in particular for producing such a type of microstructure device 20 or 40, each as a multi-electrode in array arrangement. Further details for the production of the microstructure device can be found eg in the document "Baaken et al." be removed.

In a first step 100, the planar carrier substrate is provided, for example a transparent substrate, for example, glass. In step 101, a layer of photoresist SU8 (reverse varnish) is spin coated by spin coating. In step 102, a "mask 1" is placed on this coated carrier substrate and exposed to UV light. In step 103, the exposed layer is developed, thereby selectively removing portions of the layer. In step 104, an adhesion-promoting or galvanic starting layer of chromium, gold is vapor-deposited onto this microstructure layer. In step 105, the photoresist, which had selectively shielded the carrier substrate in the case of metallic vapor deposition, is stripped off (stripping, lift-off), so that a metallic microstructure remains. This is recoated in step 106 by spin-coating. This is the first layer of the carrier substrate in which the first recesses 25, 45 (FIG. Fig. 2a and 3a ) are formed, in which the electrodes 26, 46 are introduced. This is done by placing a "Mask 2" on the photoresist coating in step 107 and exposing to UV exposure. In step 108, the photoresist coating over the chromium-gold regions is selectively removed by developing the exposed photoresist. In step 109, the chromium-gold regions are coated with silver so that the individual electrodes 26, 46 of the multi-electrode arrangement receive their silver layer 28, 48.

With SU8, layer thicknesses of, for example, 1 to 200 μm can be achieved by spin coating, which are easy to set by the viscosity of the photoresist and the rotational speeds. For the first layer, for example, at D2 = 60 microns of the electrodes in particular go up to 200 μm (depending on how much silver you want to hold). In the second layer, for example, 5-10 microns are useful to be able to fully open even small apertures during development.

For applying the second layer 24, 44 of photoresist, which ultimately defines the microapertures 31, 51, there are two alternative method embodiments 110, 115:

The step sequence 110 has proven to be very effective. In this case, in step 111, the second layer of photoresist (preferably also SU8) is spin-coated directly onto the first layer of photoresist (preferably also SU8), which already has the electrode microstructure. Surprisingly, it has been found in experiments that it is possible by the simple spin-coating of the polymer (e.g., epoxy, e.g., SU8) on the first layer of the same polymer material to apply a second layer which stably bonds to the first layer after crosslinking. But the materials of the two layers can also be different, as long as there is a sufficiently stable connection of the two layers. The "mask 3" is placed on the second layer in step 112 and UV-exposed. In step 113, the exposed photoresist is selectively removed by development so that the recesses 30, 50 are formed in the second layer, which are the second space portions of the microcavities. There is thus provided a microstructure device having a number of N microcavities (e.g., N = 4 in the figure or also, e.g., N = 16), each microcavity having an electrode.

As an alternative to the method sequence 110, the sequence 115 of method steps may be selected in order to apply the second layer with microapertures on the first layer. For this purpose, a removable sacrificial layer is applied to a separate substrate, for example a glass carrier, and a second photoresist layer (eg SU8) is spin-coated thereon. In step 117, the "mask 3" is placed on this photoresist layer and UV exposed. The exposed photoresist is developed at step 118 to selectively remove the areas for the recesses 30, 50 (microapertures). In step 119, the second layer is firmly bonded to the first layer of preferably the same photoresist in a special bonding process. This bonding process is called "thermal bonding". Two (coated) substrates are included assembled under high pressure and high temperature. In the present case 150 ° C to 200 ° C and 2000 to 4000 mbar pressure. In step 120, the sacrificial layer is dissolved and in this way the second substrate is peeled off. Thus, by alternative route 115, a microstructure device is provided which has a number of N microcavities (eg N = 4 in the figure or also eg N = 16), each microcavity having an electrode.

The present invention can provide a reliable and stable platform for high throughput electrophysiology and can be inexpensively marketed. The possibility of a high integration density and the possible excellent signal-to-noise ratios offer users considerable advantages over known devices. The end user will be able to measure all types of membrane flows quickly and with high throughput. Such measurements play an increasing role in pharmaceutical drug discovery as well as biotechnology.

Claims (8)

1. A microstructure device (1) for measuring molecular membranes (11) comprising
   a carrier substrate (2) having an upper surface (10) for supporting the membrane,
   at least one microcavity (8) of the carrier substrate for receiving an electrolyte, wherein the microcavity is upwardly open and opens out into a microaperture (9) in the upper surface of the carrier substrate,
   wherein the microaperture comprises a first characteristic diameter D1,
   at least one electrode (6) which is at least partially arranged within the microcavity and which comprises a contact side (12), that is adjoining the interior volume of the microcavity, for contacting an electrolyte,
   wherein this contact side (12) of the electrode comprises a characteristic diameter D2 which is greater than D1,
   wherein the microcavity comprises an upper space portion which is upwardly bounded by the microaperture surface and which has in the cross-section a characteristic diameter D3 and in that the microcavity comprises a lower space portion which is located below the upper space portion and which has in this cross-section a characteristic diameter D4, wherein D4>D3 and wherein the contemplated cross-section runs through the geometrical center of a planar microaperture surface and is perpendicular thereto,
   characterized in that
   the upper space portion comprises a characteristic height H3 and the lower space portion comprises a characteristic height H4, wherein H4 is less than H3.
2. A microstructure device according to claim 1, characterized in that D2 is greater than D1*1.5 (D1 multiplied by a factor c=1.5).
3. A microstructure device according claim 1 or 2, characterized in that the carrier substrate comprises a first layer (3) which is essentially located under the microaperture and within which the electrode is arranged.
4. A microstructure device according to claim 3, characterized in that the carrier substrate comprises a second layer (4) which is essentially located under the microaperture and within which the microcavity is arranged at least partially or essentially completely.
5. A microstructure device according to claim 4, characterized in that the microcavity is at least partially also arranged within the first layer.
6. A microstructure device according to at least one of the preceding claims, characterized in that the contact side comprises at least in a section or completely a microstructured or nanostructured surface or a microporous or nanoporous surface.
7. A method for producing a microstructure device according to any one of the claims 1 to 6 comprising the steps, without determining the order:

   Providing a carrier substrate;

   Applying at least one electrode onto the carrier substrate;

   Applying a layer above said carrier substrate;

   Forming the at least one microcavity by selectively removing portions of this layer, wherein these portions form the microcavity at least partially or completely.
8. A method according to claim 7, wherein this layer is directly formed on the carrier substrate or alternatively is indirectly formed on the carrier substrate by at first forming this layer on a second carrier substrate and thereafter transferring it from said second carrier substrate onto the carrier substrate.

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